L. N. Djimant

1 H NMR investigation of self-association of aromatic drug molecules in aqueous solution. Structural and thermodynamical analysis

Self-association of aromatic drug molecules, proflavine (PF), Acridine Orange (AO), ethidium bromide (EB) and actinomycin D (ActD), in aqueous salt solution has been studied by one- and two-dimensional 500 MHz 1H NMR spectroscopy. 2D-COSY and 2D-NOESY measurements were used for complete assignment of proton signals of EB and ActD in solution and for a qualitative determination of their self-association, i.e. the mutual arrangement of the drug molecules in the complexes. Concentration and temperature dependences of proton chemical shifts of the drugs have been measured. Experimental results have been analysed using indefinite non-cooperative and cooperative models of molecular self-association, enabling the determination of equilibrium reaction constants, parameters of cooperativity, the thermodynamical parameters (enthalpy and entropy) of the self-association reactions and the limiting values of drug proton chemical shifts in the complexes. The most favourable dimer structures of PF, AO, EB and ActD have been constructed using calculated values of induced chemical shifts of drug protons in conjunction with intermolecular NOEs. The results show that the drug chromophores have an antiparallel orientation in all the dimers studied.

1H-NMR determination of the thermodynamics of drug complexation with single-stranded and double-stranded oligonucleotides in solution: ethidium bromide complexation with the deoxytetranucleotides 5'-d(ApCpGpT), 5'-d(ApGpCpT), and 5'-d(TpGpCpA).

The thermodynamical parameters (free energy, enthalpy, and entropy) of complex formation between ethidium bromide and single‐stranded and double‐stranded tetranucleotides of different base sequence [5′‐d(TpGpCpA), 5′‐d(ApCpGpT), and 5′‐d(ApGpCpT) have been determined from the temperature dependencies of 500 MHz proton nmr chemical shifts. The analysis enables the contributions to be differentiated for the formation of different types of complexes (1:1, 2:1, 1:2, and 2:2) in aqueous solution. The results have been interpreted in terms of the main types of intermolecular interactions responsible for formation of the different complexes; van der Waals and electrostatic interactions are important for formation of complexes of ethidium bromide with single‐stranded tetranucleotides, whereas van der Waals and hydrophobic interactions play a significant role in the binding of the dye to the tetramer duplexes.

1H NMR Analysis of Heteroassociation of Caffeine with Mitoxanthrone in Aqueous Solution

Heteroassociation of caffeine (CAF) with the antibiotic mitoxanthrone (novatrone, NOV) in aqueous solution was studied by one-dimensional (1D) and two-dimensional (2D) 1H NMR spectroscopy (500 MHz). The concentration and temperature dependences of the proton chemical shifts of the molecules in aqueous solution have been measured. The equilibrium constants of heteroassociation between CAF and NOV and the limiting proton chemical shifts of the aromatic ligands of the associates have been determined. The most plausible structure of the 1:1 CAF–NOV heterocomplex in aqueous solution was inferred from the calculated values of the induced proton chemical shifts and quantum-mechanical screening curves for CAF and NOV. The thermodynamic parameters of the formation of the CAF–NOV heterocomplex have been calculated. The relatively high heteroassociation constant (K = 256 ± 31 M–1, T = 318 K), the positive value of entropy for heteroassociation [Δ S = 15.3 ± 4.0 J/(mole⋅K)], and the structural features of the chromophore of the novatrone molecule indicate that hydrophobic interactions play a significant role in stabilization of the CAF–NOV heterocomplex.

Structural and Thermodynamic Analysis of Daunomycin Binding with Desoxyhexanucleotides with Different Base Sequences by NMR Spectroscopy

Complexation of the anthracycline antibiotic daunomycin (DAU) with self-complementary desoxyhexanucleotides with different base sequences in the chain, 5′-d(CGTACG) and 5′-d(CGCGCG), is studied in aqueous solution. Homonuclear 2D-1H NMR spectroscopy (TOCSY and NOESY) and heteronuclear 1H–31P NMR spectroscopy (HMBC) are used for complete assignment of the nonexchangeable proton resonances and the phosphorus signals and for qualitative determination of preferable DAU binding sites, respectively. Daunomycin is preferably intercalated into the first d(CG)-site of each hexanucleotide, and the aminosaccharide ring of DAU lies in the minor groove of the hexamer duplex, partly overlapping the third base pair. Quantitative analysis of DAU complexation with desoxyhexanucleotides was done by analyzing the concentration and temperature dependences of the DAU proton chemical shifts; the equilibrium reaction constants and the thermodynamic parameters of the formation of the 1:1, 1:2, 2:1, and 2:2 DAU complexes with hexamers, as well as the limiting values of the DAU proton chemical shifts were determined in aqueous solution. The antibiotic preferably binds with the triplet sections of the nucleotide sequence containing two neighboring CG-pairs of nitrogen bases flanked by the AT-pair in 5′-d(CGTACG) compared to the triplets consisting of a sequence of three CG-pairs in the 5′-d(CGCGCG) hexamer. The specific binding of daunomycin depends on the character of the hydrophobic interactions of the aminosaccharide ring of the antibiotic in the minor groove of the DNA double helix. Calculations have been carried out to determine the most probable spatial structures of the 1:2 DAU–desoxyhexanucleotide complexes; the results agree well with X-ray diffraction data.

Complexation of the antibiotic daunomycin with desoxytetraribonucleoside triphosphate 5’-d(CpGpCpG) in aqueous solution

This paper presents the results of our study of the complexation of the antibiotic daunomycin with desoxytetranucleotide 5’-d(CpGpCpG) in aqueous salt solution by one- and two-dimensional (2D-TOCSY and 2D-NOESY)1H NMR spectroscopy (500 MHz). The concentration and temperature dependences of the proton chemical shifts of molecules were measured and used to calculate the equilibrium reaction constants, the relative contents of different types of complex as functions of concentration and temperature, the limiting values of the proton chemical shifts of daunomycin in various complexes, and the thermodynamic parameters of complexation δH and δS. It is concluded that the triplet nucleotide sequences are the preferable sites at which daunomycin is attached. The binding of the second daunomycin molecule to both single-stranded and duplex forms of tetramer is markedly anticooperative. This is explained by the presence in the antibiotic of a positively charged amino sugar residue creating steric hindrances for the attachment of the second antibiotic molecule to the short tetranucleotide sequence. The most plausible spatial structure of the 1:2 complex of antibiotic with desoxytetranucleotide is constructed using the calculated values of the induced proton chemical shifts of daunomycin and 2D-NOE data.

Analysis of the interaction of ethidium bromide with a DNA octamer 5’-d(GpApCpApTpGpTpC) in aqueous solution using1H NMR data

Complexation of a phenanthridine dye ethidium bromide with a desoxyoligonucleotide 5’-d(GpApCpAp-TpGpTpC) in aqueous salt solution is studied by one- and two-dimensional1H NMR spectroscopy. Two-dimensional correlated homonuclear PMR spectroscopy (2D-TOCSY and 2D-N0ESY) was used for complete assignment of the proton signals of molecules in solution and for qualitative analysis of the character of interaction between ethidium bromide and desoxyoctanucleotide. The concentration dependences of the proton chemical shifts of the molecules were measured at three temperatures (T1 = 298 K, T2 = 308 K, and T3 = 318 K); the temperature dependences were measured in the temperature range 278–358 K. Different schemes of dye complexation with an octamer duplex involving different molecular associates in solution are considered. The equilibrium constants of the reactions, the corresponding thermodynamic parameters (δH0, δS0), and the limiting values of the chemical shifts of ethidium bromide protons in the complexes are determined. The relative contents of complexes of different types in solution (dye complexes with desoxyoctanucleotide in duplex form) are analyzed, and peculiarities of the dynamic equilibrium depending on the ratio of dye and octamer concentrations and temperature are established. The most probable structures of the 1:2 and 2:2 intercalated complexes corresponding to dye intercalation into the pyrimidine-purine sites of the desoxyoctanucleotide duplex are derived using the calculated values of the induced proton chemical shifts of ethidium bromide and two-dimensional PMR data.

Structural analysis of the complex of phenoxazone antibiotic actinocyl-bis(2-dimethylaminoethyl)amide with deoxytetranucleotide 5′-d(TpGpCpA) by two-dimensional 1H-NMR spectroscopy and molecular mechanics

The spatial structure of an intercalated complex of the synthetic phenoxazone antibiotic actinocyl-bis-(2-dimethylaminoethyl)amide (ActII) and the self-complementary deoxytetranucleotide 5′-d(TpGpCpA) was studied using two-dimensional NMR (2D-NOESY) spectroscopy of the complex in an aqueous solution and molecular mechanics simulation. Distinctive features of the conformation of ActII-DNA complexes were determined for two possible orientations of the ActII chromophore at the intercalation site.